Protonated species are often postulated as key intermediates in enzyme-induced, as well as simple chemical reactions. In this research project a simple technique of considerable generality is explored which, when applied to biological reactions, can be used to establish not only the suspected protonated species, but also the specific site of protonation. Irreversibly alkylated, charged analogs of the suspected intermediates are simply synthesized, and then tested for the desired reactivity. The coenzyme, biotin, plays a pivotal role in CO2 fixation reactions, for example in the conversion of acetyl CoA to malonyl CoA. There is considerable controversy concerning the exact nature of the CO2-transfer agent. Using "irreversible acids" we are attempting to ascertain whether or not protonated forms of biotin are needed for this transfer. We have recently prepared both N-carbomethyoxy-2-phenacyl thioimidazoline and its N-methyl salt, N-methyl-N-carbomethoxy-2-phenacylthiotimidazolinium fluoroborate. Both substrates can be considered potential models for carboxy-biotin complexes. The reactivity of each of these substrates toward base has been evaluated. Successful transfer of the carbomethoxyl occurs only in the charged analog, giving 2-(1' -methyl-2' -imidazolin-2' -thiyl)-methyl benzoylacetate. These results indicate that CO2 transfer to an acceptor molecule should be facilitated by prior protonation of the biotin-CO2 intermediate. Two mechanisms are currently being investigated that satisfy this criteria.